Krill oil and carotenoid composition, associated method and delivery system

ABSTRACT

A medicine delivery system includes an inner capsule containing carotenoids and an outer capsule in which the inner capsule is contained within the outer capsule and the outer capsule containing a therapeutically effective amount of krill oil. In one example, the carotenoids comprise at least S,S′-astaxanthin derived from  Haematococcus pluvialis , and one or more of lutein and/or trans-zeaxanthin or meso-zeaxanthin. The medicine delivery system also includes 0.5 to 8 mg of astaxanthin, 2 to 15 mg of lutein and 0.2 to 12 mg of trans-zeaxanthin contained within the inner capsule. In a specific example, the medicine delivery system includes about 4 mg of astaxanthin, about 10 mg of lutein and about 1.2 mg of trans-zeaxanthin contained within the inner capsule.

RELATED APPLICATION(S)

This application is a continuation-in-part of U.S. patent applicationSer. No. 12/840,396 filed Jul. 21, 2010, which is based on provisionalpatent application Ser. No. 61/227,881, filed Jul. 23, 2009.

FIELD OF THE INVENTION

The present invention relates to a krill oil and carotenoid compositionused for eye care or other medical applications and medicine deliverysystem for the composition. The present invention also relates to amethod of preventing, retarding and ameliorating central nervous systemand eye diseases, including treating eye insult resulting from diseaseor injury, such as age-related macular degeneration, photic injury,photoreceptor cell or ganglion cell damage, ischemic insult-relateddiseases, cataracts, dry eye syndromes and inflammatory diseases.

BACKGROUND OF THE INVENTION

The eye is an extension of the brain, and therefore a part of thecentral nervous system. Accordingly, in the case of an eye injury ordisease, i.e., a retinal injury or disease, the diseases are oftenwithout treatment and the eye cannot be transplanted. Eye diseases andinjuries that presently are untreatable include retinal photic injury,retinal ischemia-induced eye injury, age-related macular degeneration,and other eye diseases and injuries that are induced by singlet oxygenand other free radical species.

It has been hypothesized that a major cause of these untreatable retinaland other eye diseases and injuries is the generation and presence ofsinglet oxygen and other free radical species. Singlet oxygen and freeradical species can be generated by a combination of light, oxygen,other reactive oxygen species like hydrogen peroxide, superoxide orduring reperfusion after an ischemic insult resulting in highly reactiveNOx release.

The eye is subjected to continuous light exposure because the primarypurpose of the eye is light perception. Therefore, some untreatablediseases and injuries to the eye result from the continuous exposure ofthe eye to light, coupled with the highly-oxygenated environment in theeye.

The process of light perception is initiated in the photoreceptor cells.The photoreceptor cells are a constituent of the outer neuronal layer ofthe retina, which is a component of the central nervous system. Thephotoreceptor cells are well sheltered in the center of the eye, and areprotected structurally by the sclera, nourished by thehighly-vascularized uvea and safeguarded by the blood-retinal barrier ofthe retinal pigmented epithelium.

The primary function of the photoreceptor cells is to convert light intoa physic-chemical signal (transduction) and to transmit this signal tothe other neurons (transmission). During the transduction andtransmission processes, the metabolic activities of these neurons arechanged dramatically. Even though the photoreceptor cells are securelyprotected in the interior of the eye, these cells are readily accessibleto light because their primary function is light detection. Excessivelight energy reaching the retina can cause damage to these neurons,either directly or indirectly, by overwhelming the metabolic systems ofthese cells.

The combination of continuous and/or excessive exposure to light, andthe relatively high concentration of oxygen in the eye, generatessinglet oxygen and other free radical species. Singlet oxygen and freeradical species also can be generated by enzymatic processes independentfrom light exposure. The free radical species and singlet oxygen arereactive entities that can oxidize polyunsaturated fatty acids. Theretina contains the highest concentration of polyunsaturated fatty acidsof any tissue in the human body, and per-oxidation of thepolyunsaturated fatty acids in cell membranes of the retina by hydroxylradicals (OH) or superoxide (O₂) radicals can propagate additional freeradical species. These free radical species can lead to functionalimpairment of the cell membranes and cause temporary or permanent damageto retinal tissue. It has been theorized that the generation of singletoxygen and free radical species therefore underlies the pathogenesis oflight-induced retinopathy and post-ischemic reflow injury. In addition,a deficiency in removing these reactive free radical species can alsocontribute to various diseases of the eye.

A number of natural mechanisms protect the photoreceptor cells fromlight injury. For example, the ocular media, including the cornea,aqueous, lens, and vitreous, filter most of the light in the ultravioletregion. However, after cataract extraction or other surgicalintervention, some of these protective barriers are removed ordisturbed, whereby the photoreceptor cells are more susceptible todamage by radiant energy. The photoreceptor cells also possess otherforms of protection from photic injury, for example, the presence ofantioxidant compounds to counteract the free radical species generatedby light. As will be demonstrated hereafter, antioxidants, which quenchand/or scavenge singlet oxygen, hydrogen peroxide, superoxide andradical species, minimize injury to the photoreceptor cells. Themost-important area of the retina where such protection is necessary isthe fovea or central region of the macula. Even though severalprotective mechanisms are present in the eye, a leading cause ofblindness in the United States is age-related photoreceptordegeneration. Clinically, photoreceptor degeneration, as seen inage-related macular degeneration, is causally related to excessiveexposure to high energy UVA and UVB ultraviolet light. The causes ofage-related macular degeneration, which is characterized by a loss ofphotoreceptor neurons resulting in decreased vision, are still beinginvestigated. Epidemiological studies indicate that age-relatedphotoreceptor degeneration, or age-related macular degeneration, isrelated to several factors including age, sex, family history, color ofthe iris, nutritional deficiency, immunologic disorders, cardiovascularand respiratory diseases and pre-existing eye diseases. Advancing age isthe most significant factor. Recently, it has been demonstrated thataging eyes have a decreased amount of carotenoids deposited on theretina. Clinical and laboratory studies indicate that photic injury isat least one cause of age-related macular degeneration because of thecumulative effect of repeated mild photic insult which leads to agradual loss of photoreceptor cells.

Age-related macular degeneration is an irreversible blinding disease ofthe retina. Unlike cataracts which can be restored by replacing thediseased lens, age-related macular degeneration cannot be treated byreplacing the diseased retina because the retina is a component of thecentral nervous system. Therefore, because no treatment for this diseaseexists once the photoreceptors are destroyed, prevention is the only wayto address age-related macular degeneration. Presently, prevention ofage-related macular degeneration resides in limiting or preventing lightand oxygen-induced (i.e., free radical-induced) damage to the retinabecause the retina is the only organ that is continuously exposed tohigh levels of light in a highly-oxygenated environment.

In addition to photic injury, eye injury and disease can result fromsinglet oxygen and free radical species generated during reperfusionafter an ischemic insult. Ischemic insult to retinal ganglion cells andto neurons of the inner layers of retina causes loss of vision. Loss ofvision accompanies diabetic retinopathy, retinal arterial occlusion,retinal venous occlusion and glaucoma, each of which insults the eyedepriving the eye of oxygen and nutrition via ischemic insult.

The damage to the retinal ganglion cells has been attributed toischemia, and subsequent reperfusion during which free radicals aregenerated.

The pathogenesis of photic injury, of age-related macular degeneration,of ischemia/reperfusion damage, of traumatic injury and of inflammationsof the eye have been attributed to singlet oxygen and free radicalgeneration, and subsequent free radical-initiated reactions.Investigators therefore studied the role of antioxidants in preventingor ameliorating these diseases and injuries of the central nervoussystem in general, and the eye in particular.

For example, ascorbate was investigated as an agent to treat retinalphotic injury. Ascorbate is a reducing agent which is present in theretina in a high concentration. Studies indicated that ascorbate in theretina can act as an antioxidant and is oxidized by free radical speciesgenerated during excessive light exposure.

Administration of ascorbate reduced the loss of rhodopsin after photicexposure, thereby suggesting that ascorbate offered protection againstretinal photic injury. A decrease in rhodopsin levels is an indicator ofphotic eye injury. The protective effect of ascorbate is dose-dependent,and ascorbate was effective when administered before light exposure.Morphometric studies of the photoreceptor nuclei remaining in the retinaafter light exposure showed that rats given ascorbate supplements hadsubstantially less retinal damage. Morphologically, rats with ascorbatesupplements also showed better preservation of retinal pigmentedepithelium.

The above studies led to the hypothesis that ascorbate mitigates retinalphotic injury because of its antioxidant properties, which areattributed to its redox properties. Ascorbate is a scavenger ofsuperoxide radicals and hydroxy radicals and also quenches singletoxygen, all of which are formed during retinal photic injury. Thishypothesis accounts for the presence of high levels ofnaturally-occurring ascorbate in a normal retina.

Therefore, antioxidants which inhibit free radical formation, or whichquench singlet oxygen and scavenge free radical species, can decreaselipid per-oxidation and ameliorate photic injury andischemic/reperfusion injury in the retina. Antioxidants originally wereinvestigated because they are known constituents of human tissue.However, antioxidants that are not naturally occurring in human tissuewere also tested. In particular, in addition to ascorbate, antioxidantssuch as 2,6-di-tert-butylphenol, gamma-oryzanol, alpha-tocopherol,mannitol, reduced glutathione, and various carotenoids, includinglutein, zeaxanthin and astaxanthin have been studied for an ability tocomparatively quench singlet oxygen and scavenge free radical species invitro. These and other antioxidants have been shown in vitro to beeffective quenchers and scavengers for singlet oxygen and free radicals.In particular, the carotenoids, as a class of compounds, are veryeffective singlet oxygen quenchers and free radical scavengers. However,individual carotenoids differ in their ability to quench singlet oxygenand scavenge for free radical species.

The carotenoids are naturally-occurring compounds that have antioxidantproperties. The carotenoids are common compounds manufactured by plants,and contribute greatly to the coloring of plants and some animals. Anumber of animals, including mammals, are unable to synthesizecarotenoids de novo and accordingly rely upon diet to provide carotenoidrequirements. Mammals also have a limited ability to modify carotenoids.A mammal can convert beta-carotene to vitamin A, but most othercarotenoids are deposited in mammalian tissue in unchanged form.

With respect to humans, about ten carotenoids are found in human serum.The major carotenoids in human serum are beta-carotene, alpha-carotene,cryptoxanthin, lycopene and lutein. Small amounts of zeaxanthin,phytofluene, and phytoene are found in human organs. However, of the tencarotenoids found in human serum, only two, trans- and/ormeso-zeaxanthin and lutein, have been found in the human retina.Zeaxanthin is the predominant carotenoid in the central macula or fovealregion and is concentrated in the cone cells in the center of theretina, i.e., the fovea. Lutein is predominantly located in theperipheral retina in the rod cells. Therefore, the eye preferentiallyassimilates zeaxanthin over lutein in the central macula which is a moreeffective singlet oxygen scavenger than lutein. It has been theorizedthat zeaxanthin and lutein are concentrated in the retina because oftheir ability to quench singlet oxygen and scavenge free radicals, andthereby limit or prevent photic damage to the retina.

Therefore only two of the about ten carotenoids present in human serumare found in the retina. Beta-carotene and lycopene, the two mostabundant carotenoids in human serum, either have not been detected orhave been detected only in minor amounts in the retina. Beta-carotene isrelatively inaccessible to the retina because beta-carotene is unable tocross the blood-retinal brain barrier of the retinal pigmentedepithelium effectively. It also is known that another carotenoid,canthaxanthin, can cross the blood-retinal brain barrier and reach theretina. Canthaxanthin, like all carotenoids, is a pigment and candiscolor the skin. Canthaxanthin provides a skin color that approximatesa suntan, and accordingly has been used by humans to generate anartificial suntan. However, an undesirable side effect in individualsthat ingested canthaxanthin at high doses for an extended time was theformation of crystalline canthaxanthin deposits in the inner layers ofthe retina. Therefore, the blood-retinal brain barrier of the retinalpigmented epithelium permits only particular carotenoids to enter theretina. The carotenoids other than zeaxanthin and lutein that do enterthe retina cause adverse effects, such as the formation of crystallinedeposits by canthaxanthin, which may take several years to dissolve.Canthaxanthin in the retina also caused a decreased adaptation to thedark.

Investigators have unsuccessfully sought additional antioxidants tofurther counteract the adverse affects of singlet oxygen and freeradical species on in the eye. The investigators have studied theantioxidant capabilities of several compounds, including variouscarotenoids. Even though the carotenoids are strong antioxidants,investigators have failed to find particular carotenoids among the 600naturally-occurring carotenoids that effectively quench singlet oxygenand scavenge for free radical species, that are capable of crossing theblood-retinal brain barrier, that do not exhibit the adverse affects ofcanthaxanthin after crossing the blood-retinal brain barrier, and thatameliorate eye disease or injury and/or retard the progression of adegenerative disease of the eye and are more potent anti-oxidants thaneither lutein or zeaxanthin.

Many scientific papers are directed to eye diseases and injuries, suchas age-related macular degeneration, causes of the damage resulting fromthe diseases or injuries, and attempts to prevent or treat such diseasesand injuries. The publications, which discuss various antioxidants,including the carotenoids and other antioxidants like alpha-tocopherol,include:

M. O. M. Tso, “Experiments on Visual Cells by Nature and Man: In Searchof Treatment for Photoreceptor Degeneration,” InvestigativeOphthalmology and Visual Science, 30(12), pp. 2421-2454 (December,1989);

W. Schalch, “Carotenoids in the Retina—A Review of Their Possible Rolein Preventing or Limiting Damage Caused by Light and Oxygen,” FreeRadicals and Aging, I. Emerit et al. (ed.), Birkhauser Verlag, pp.280-298 (1992);

M. O. M. Tso, “Pathogenetic Factors of Aging Macular Degeneration,”Ophthalmology, 92(5), pp. 628-635 (1985);

M. Mathews-Roth, “Recent Progress in the Medical Applications ofCarotenoids,” Pure and Appl. Chem., 63(1), pp. 147-156 (1991);

W. Miki, “Biological Functions and Activities of Animal Carotenoids,”Pure and Appl. Chem., 63(1), pp. 141-146 (1991);

M. Mathews-Roth, “Carotenoids and Cancer Prevention-Experimental andEpidemiological Studies,” Pure and Appl. Chem., 57(5), pp. 717-722(1985);

M. Mathews-Roth, “Porphyrin Photosensitization and Carotenoid Protectionin Mice; In Vitro and In Vivo Studies,” Photochemistry and Photobiology,40(1), pp. 63-67 (1984);

P. DiMascio et al., “Carotenoids, Tocopherols and Thiols as BiologicalSinglet Molecular Oxygen Quenchers,” Biochemical Society Transactions,18, pp. 1054-1056 (1990);

T. Hiramitsu et al., “Preventative Effect of Antioxidants on LipidPeroxidation in the Retina,” Ophthalmic Res., 23, pp. 196-203 (1991);

D. Yu et al., “Amelioration of Retinal Photic Injury by Beta-Carotene,”ARVO Abstracts Invest. Ophthalmol. Vis. Sci., 28 (Suppl.), p. 7, (1987);

M. Kurashige et al., “Inhibition of Oxidative Injury of BiologicalMembranes by Astaxanthin,” Physiol. Chem. Phys. and Med. NMR, 22, pp.27-38 (1990); and

N. I. Krinsky et al., “Interaction of Oxygen and Oxy-radicals WithCarotenoids,” J. Natl. Cancer Inst., 69(1), pp. 205-210 (1982).

Anon., “Bio & High Technology Announcement Itaro,” Itaro RefrigeratedFood Co., Ltd.

Anon., “Natural Astaxanthin & Krill Lecithin,” Itaro Refrigerated FoodCo., Ltd.

Johnson, E. A. et al., “Simple Method for the Isolation of Astaxanthinfrom the Basidomycetous Yeast Phaffia rhodozyma,” App. Environ.Microbiol., 35(6), pp. 1155-1159 (1978).

Kirschfeld, K., “Carotenoid Pigments: Their Possible Role in ProtectingAgainst Photooxidation in Eyes and Photoreceptor Cells,” Proc. R. Soc.Land., B216, pp. 71-85 (1982).

Latscha, T., “Carotenoids-Carotenoids in Animal Nutrition,”Hoffmann-LaRoche Ltd., Basel, Switzerland.

Li, Z. et al., “Desferrioxime Ameliorated Retinal Photic Injury inAlbino Rats,” Current Eye Res., 10(2), pp. 133-144 (1991).

Mathews-Roth, M., “Porphyrin Photosensitization and CarotenoidProtection in Mice; In Vitro and In Vivo Studies,” Photochemistry andPhotobiology, 40(1), pp. 63-67 (1984).

Michon, J. J. et al., “A Comparative Study of Methods of PhotoreceptorMorphometry,” Invest. Ophthalmol. Vis. Sci., 32, pp. 280-284 (1991).

Tso, M. O. M., “Pathogenetic Factors of Aging Mascular Degeneration,”Ophthalmology, 92(5), pp. 628-635 (1985).

Yu, D. et al., “Amelioration of Retinal Photic Injury by Beta-Carotene,”ARVO Abstracts Invest. Ophthalmol. Vis. Sci., 28 (Suppl.), p. 7, (1987).

In general, the above-identified publications support the hypothesisthat singlet oxygen and free radical species are significantcontributors to central nervous system, and particularly eye injury anddisease. For example, it has reported that consumption of anantioxidant, such as ascorbic acid (Vitamin C), alpha-tocopherol(Vitamin E) or beta-carotene (which is converted in vivo to lutein), candecrease the prevalence of age-related macular degeneration.

The above-identified publications also demonstrated that severalcarotenoids, including astaxanthin, are strong antioxidants compared tobeta-carotene, ascorbic acid and other widely used antioxidants invitro. The publications also relate that (1) only particular carotenoidsselectively cross the blood-retinal brain barrier, and that (2) certaincarotenoids other than zeaxanthin and lutein that cross theblood-retinal brain barrier cause adverse affects.

In general, the above-identified publications teach that astaxanthin isa more effective antioxidant than carotenoids such as zeaxanthin,lutein, tunaxanthin, canthaxanthin, beta-carotene, and alpha-tocopherolin vitro. For example, the in vitro and in vivo studies disclosed in theKurashige et al. publication with respect to astaxanthin demonstratedthat the mean effective concentration of astaxanthin which inhibitslipid peroxidation was 500 times lower than that of alpha-tocopherol.Similarly, the Miki publication discloses that, in vitro, astaxanthinexhibits a strong quenching effect against singlet oxygen and a strongscavenging effect against free radical species.

This free radical theory of retinal damage has been advanced byinvestigators examining the effectiveness of various antioxidants inameliorating these diseases.

To date, investigative efforts have been directed to preventing diseasesand injury because the resulting free radical-induced damage is noteffectively treatable. Therefore, a need exists for a method not only toprevent or retard, but also to ameliorate, degenerative and traumaticdiseases and injuries to the central nervous system, and particularlythe eye. The copending '396 parent application discloses atherapeutically effective amount of a synergistic multi-ingredientcomposition of mixed carotenoids comprising at least S,S′-astaxanthinderived from Haematococcus pluvialis, and one or more of lutein and/ortrans-zeaxanthin or meso-zeaxanthin admixed with a therapeuticallyeffective amount of krill oil containing phospholipid bound andtriglyceride bound EPA and DHA in which said krill oil contains at least30% total phospholipids. The composition includes 50 to 1000 mg of krilloil, 0.5 to 8 mg of astaxanthin, 2 to 15 mg of lutein and 0.2 to 12 mgof trans-zeaxanthin.

Unexpectedly, it has been found that the addition of carotenoids andespecially astaxanthin to krill oil results in an apparent chemicalreaction between the two components with the possibletrans-esterification occurring between the krill oil fatty acid estersand partially esterified carotenoids and creating a new compound.Therefore, a delivery mechanism is beneficial for the composition toprevent the disappearance of carotenoids. The “reacted” carotenoidscould also be beneficial in an associated method of treating andcomposition.

SUMMARY OF THE INVENTION

A medicine delivery system includes an inner capsule containingcarotenoids and an outer capsule in which the inner capsule is containedwithin the outer capsule and the outer capsule containing atherapeutically effective amount of krill oil. In one example, thecarotenoids comprise at least S,S′-astaxanthin derived fromHaematococcus pluvialis, and one or more of lutein and/ortrans-zeaxanthin or meso-zeaxanthin. The medicine delivery system alsoincludes 0.5 to 8 mg of astaxanthin, 2 to 15 mg of lutein and 0.2 to 12mg of trans-zeaxanthin contained within the inner capsule. In a specificexample, the medicine delivery system includes about 4 mg ofastaxanthin, about 10 mg of lutein and about 1.2 mg of trans-zeaxanthincontained within the inner capsule.

In another example, the krill oil contains phospholipid bound andtriglyceride bound EPA and DHA and the krill oil contains at least 30%total phospholipids. In another example, the krill oil is about 50 toabout 1,000 mg contained in the outer capsule.

A method of treating an individual is also set forth by administering atherapeutically effective amount of synergistic multi-ingredientcomposition of krill oil and carotenoids and reacting to carotenoids invivo with the krill oil. In an example, the method includesadministering carotenoids comprising at least S,S′-astaxanthin derivedfrom Haematococcus pluvialis and one or more of lutein and/ortrans-zeaxanthin or meso-zeaxanthin. In another example, the methodincludes administering 0.5 to 8 mg of astaxanthin, 2 to 15 mg of luteinand 0.2 to 12 mg of trans-zeaxanthin. About 4 mg of astaxanthin, about10 mg of lutein and about 1.2 mg of trans-zeaxanthin can beadministered. The krill oil in yet another example contains phospholipidbound and triglyceride bound EPA and DHA and about 30% totalphospholipids. About 50 to 1,000 mg of krill oil can be administered.

A composition is disclosed that includes a synergistic multi-ingredientcomposition of krill oil containing phospholipid bound and triglyceridebound EPA and DHA in which the krill oil contains at least 30% totalphospholipids and mixed carotenoids comprising at least S,S′-astaxanthinderived from Haematococcus pluvialis that are reacted with the krilloil, and one or more of lutein and/or trans-zeaxanthin ormeso-zeaxanthin.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects, features and advantages of the present invention willbecome apparent from the detailed description of the invention whichfollows, when considered in light of the accompanying drawings in which:

FIG. 1 is an example of a medicine delivery system that includes aninner capsule containing carotenoids and an outer capsule containingkrill oil in accordance with a non-limiting example.

FIG. 2 is chart showing stability data of various formulation blends ofthe composition of carotenoids and krill oil in accordance with anon-limiting example.

FIGS. 3 and 4 show stability analytical charts for various formulationblends of the composition of carotenoids and krill oil at respective 20degrees C. and 50 degrees C. in accordance with a non-limiting example.

FIG. 5 is a chart showing a summary of changes by ultraviolet radiationof the astaxanthin in the composition in accordance with a non-limitingexample.

FIG. 6 is another chart showing astaxanthin stability for thecomposition at 20 degrees C. and 50 degrees C.

FIG. 7 are chemical formula of various carotenoids and showing thedifferences among them.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will now be described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein. Rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art.

The leading cause of visual loss among elderly persons is dry or atropicAMD, which has an increasingly important social and economic impact inthe United States. As the size of the elderly population increases inthis country, AMD will become a more prevalent cause of blindness thanboth diabetic retinopathy and glaucoma combined. Although lasertreatment has been shown to reduce the risk of extensive macularscarring from the “wet” or neovascular form of the disease, there arecurrently no effective treatments for the vast majority of patients withwet AMD.

The Eye Diseases Prevalence Research Group (EDPRG) attributes AMD as themajor cause of blindness among elderly people of European ancestry.Among white persons, AMD is believed to account for more than 50% of allblinding conditions.

The EDPRG estimates that approximately 1.2 million residents of the USare living with neovascular AMD and 970,000 are living with geographicatrophy, while 3.6 million are living with bilateral large drusen. Inthe next 20 years these values are expected to increase by 50% withprojected demographic shifts.

Age-related developmental changes in retinal morphology and energymetabolism, as well as cumulative effects of environmental exposures mayrender the neural and vascular retina and retinal pigment epitheliummore susceptible to damage in late adulthood. Along with these metabolicand structural changes and exposures, the aging eye also experiences areduction in the potency of endogenous and exogenous defense systems.Pharmacological and surgical treatment options are of limited scope andefficacy currently. They are costly and may result in complications assevere as end-stage disease. The likelihood of vision loss among personswith neovascular AMD can be reduced with anti-VEGF treatment,photodynamic therapy, and laser photocoagulation.

Nutrient-based preventative treatments for AMD development andprogression have been examined in several studies including AREDSI, aNEI-sponsored study, the LAST, TOZAL and CARMIS studies for example.AREDS was a multi-center study of the natural history of AMD andcataract. AREDS included a controlled randomized clinical trial designedto evaluate the effect of pharmacological doses of zinc and/or aformulation containing nutrients with antioxidant properties (vitamin C,vitamin E, and β-carotene) on the rate of progression to advanced AMDand on visual acuity outcomes. The use of the combination ofantioxidants and zinc reduced the risk of development of advanced AMD inparticipants who had at least a moderate risk of developing AMD by about25%. The overall risk of moderate vision loss [≧15 letters on the EarlyTreatment Diabetic Retinopathy Study (ETDRS) chart] was reduced by 19%at 5 years.

Of approximately 600 carotenoids identified in nature in the human diet,and 20 in human serum, only two forms of dietary xanthophylls, luteinand zeaxanthin, are present in human macular pigment. Lutein representsapproximately 36% of all retinal carotenoids; zeaxanthin andmeso-zeaxanthin each represent about 18%.

The natural tissue distribution, biochemical, and biophysicalcharacteristics of lutein provide a reasonable basis for speculatingthat this nutrient acts in biological systems as: (1) an importantstructural molecule within cell membranes; (2) a short-wavelength lightfilter; (3) a modulator of intra- and extracellular reduction-oxidation(redox) balance; and (4) a modulator in signal transduction pathways.Lutein and zeaxanthin were considered for inclusion in the AREDSformulation; however, at the time of AREDS' initiation, neithercarotenoid was readily available for manufacturing in a researchformulation.

The evidence base suggests that macular xanthophylls in combination withomega-3 LCPUFAs from fish oil may act as modifiable factors capable ofmodulating processes implicated in existing AMD pathogenesis andprogression and is the basis for the on-going US Government sponsoredAREDS II study. Intake of these compounds may also show merit as awell-tolerated preventive intervention. Biochemical and biophysicalproperties of these compounds demonstrate a capacity to modulate factorsand processes that activate and are activated by exposures associatedwith aging. These exposures include developmental changes associatedwith aging, chronic light exposure, alterations in energy metabolism,and cellular signaling pathways.

Dry Eye Syndrome

According to C Stephen Foster, MD, FACS, FACR, FAAO, Clinical Professorof Ophthalmology, Harvard Medical School; Consulting Staff, Departmentof Ophthalmology, Massachusetts Eye and Ear Infirmary; Founder andPresident, Ocular Immunology and Uveitis Foundation, Massachusetts EyeResearch and Surgery Institution et al' dry eye is a very commondisorder affecting a significant percentage (approximately 10-30%) ofthe population, especially those older than 40 years.

In the United States, an estimated 3.23 million women and 1.68 millionmen, a total of 4.91 million people, aged 50 years and older areaffected.

Dry eye is a multi-factorial disease of the tears and the ocular surfacethat results in symptoms of discomfort, visual disturbance, and tearfilm instability with potential damage to the ocular surface. Dry eye isaccompanied by increased osmolarity of the tear film and inflammation ofthe ocular surface.

The tear layer covers the normal ocular surface. Generally, it isaccepted that the tear film is made up of 3 intertwined layers, asfollows:

1) A superficial thin lipid layer (0.11 μm) is produced by the meibomianglands, and its principal function is to retard tear evaporation and toassist in uniform tear spreading.

2) A middle thick aqueous layer (7 μm) is produced by the main lacrimalglands (reflex tearing), as well as the accessory lacrimal glands ofKrause and Wolfring (basic tearing).

3) An innermost hydrophilic mucin layer (0.02-0.05 μm) is produced byboth the conjunctiva goblet cells and the ocular surface epithelium andassociates itself with the ocular surface via its loose attachments tothe glycocalyx of the microplicae of the epithelium. It is thehydrophilic quality of the mucin that allows the aqueous to spread overthe corneal epithelium.

The lipid layer produced by the meibomian glands acts as a surfactant,as well as an aqueous barrier (retarding evaporation of the underlyingaqueous layer), and provides a smooth optical surface. It may also actas a barrier against foreign particles and may also have someantimicrobial properties. The glands are holocrine in nature, and so thesecretions contain both polar lipids (aqueous-lipid interface) andnonpolar lipids (air-tear interface) as well as proteinaceous material.All of these are held together by ionic bonds, hydrogen bonds, and vander Waals forces. The secretions are subject to neuronal(parasympathetic, sympathetic, and sensory sources), hormonal (androgenand estrogen receptors), and vascular regulation. Evaporative loss ispredominantly due to meibomian gland dysfunction (MGD).

The aqueous component is produced by the lacrimal glands. This componentincludes about 60 different proteins, electrolytes, and water. Lysozymeis the most abundant (20-40% of total protein) and also the mostalkaline protein present in tears. It is a glycolytic enzyme that iscapable of breaking down bacterial cell walls. Lactoferrin hasantibacterial and antioxidant functions, and the epidermal growth factor(EGF) plays a role in maintaining the normal ocular surface and inpromoting corneal wound healing. Albumin, transferrin, immunoglobulin A(IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) are alsopresent.

Aqueous tear deficiency (ATD) is the most common cause of dry eye, andit is due to insufficient tear production. The secretion of the lacrimalgland is controlled by a neural reflex arc, with afferent nerves(trigeminal sensory fibers) in the cornea and the conjunctiva passing tothe pons (superior salivary nucleus), from which efferent fibers pass,in the nervus intermedius, to the pterygopalatine ganglion andpostganglionic sympathetic and parasympathetic nerves terminating in thelacrimal glands.

Keratoconjunctivitis sicca (KCS) is the name given to this ocularsurface disorder. KCS is subdivided into Sjogren syndrome (SS)associated KCS and non-SS associated KCS. Patients with aqueous teardeficiency have SS if they have associated xerostomia and/or connectivetissue disease. Patients with primary SS have evidence of a systemicautoimmune disease as manifested by the presence of serumauto-antibodies and very severe aqueous tear deficiency and ocularsurface disease. These patients, mostly women, do not have a separate,identifiable connective tissue disease. Subsets of patients with primarySS lack evidence of systemic immune dysfunction, but they have similarclinical ocular presentation. Secondary SS is defined as KCS associatedwith a diagnosable connective tissue disease, most commonly rheumatoidarthritis but also SLE and systemic sclerosis.

Non-SS KCS is mostly found in postmenopausal women, in women who arepregnant, in women who are taking oral contraceptives, or in women whoare on hormone replacement therapy (especially estrogen only pills). Thecommon denominator here is a decrease in androgens, either from reducedovarian function in the postmenopausal female or from increased levelsof the sex hormone binding globulin in pregnancy and birth control pilluse. Androgens are believed to be trophic for the lacrimal and meibomianglands. They also exert potent anti-inflammatory activity through theproduction of transforming growth factor beta (TGF-beta), suppressinglymphocytic infiltration.

Lipocalins (previously known as tear-specific prealbumin), which arepresent in the mucous layer, are inducible lipid-binding proteinsproduced by the lacrimal glands that lower the surface tension of normaltears. This provides stability to the tear film and also explains theincrease in surface tension that is seen in dry eye syndromescharacterized by lacrimal gland deficiency. Lipocalin deficiency canlead to the precipitation in the tear film, forming the characteristicmucous strands seen in patients with dry eye symptomatology.

The glycocalyx of the corneal epithelium contains the transmembranemucins (glycosylated glycoproteins present in the glycocalyx) MUC1,MUC4, and MUC16. These membrane mucins interact with soluble, secreted,gel-forming mucins produced by the goblet cells (MUC5AC) and also withothers like MUC2. The lacrimal gland also secretes MUC7 into the tearfilm.

These soluble mucins move about freely in the tear film (a processfacilitated by blinking and electrostatic repulsion from the negativelycharged transmembrane mucins), functioning as clean-up proteins (pickingup dirt, debris, and pathogens), holding fluids because of theirhydrophilic nature, and harboring defense molecules produced by thelacrimal gland. Transmembrane mucins prevent pathogen adherence (andentrance) and provide a smooth lubricating surface, allowing lidepithelia to glide over corneal epithelia with minimal friction duringblinking and other eye movements. Recently, it has been suggested thatthe mucins are mixed throughout the aqueous layer of tears (owing totheir hydrophilic nature) and, being soluble, move freely within thislayer.

Mucin deficiency (caused by damage to the goblet cells or the epithelialglycocalyx), as seen in Stevens-Johnson syndrome or after a chemicalburn, leads to poor wetting of the corneal surface with subsequentdesiccation and epithelial damage, even in the presence of adequateaqueous tear production.

Pathophysiology

A genetic predisposition in SS associated KOS exists as evident by thehigh prevalence of human leukocyte antigen B8 (HLA-B8) haplotype inthese patients. This condition leads to a chronic inflammatory state,with the production of auto-antibodies, including antinuclear antibody(ANA), rheumatoid factor, fodrin (a cytoskeletal protein), themuscarinic M3 receptor, or SS-specific antibodies (eg, anti-RO [SS-A],anti-LA [SS-B]), inflammatory cytokine release, and focal lymphocyticinfiltration (ie, mainly CD4⁺ T cells but also B cells) of the lacrimaland salivary gland, with glandular degeneration and induction ofapoptosis in the conjunctiva and lacrimal glands. This results indysfunction of the lacrimal gland, with reduced tear production, andloss of response to nerve stimulation and less reflex tearing. Active Tlymphocytic infiltrate in the conjunctiva also has been reported innon-SS associated KCS.

Both androgen and estrogen receptors are located in the lacrimal andmeibomian glands. SS is more common in postmenopausal women. Atmenopause, a decrease in circulating sex hormones (ie, estrogen,androgen) occurs, possibly affecting the functional and secretory aspectof the lacrimal gland. Forty years ago, initial interest in this areacentered on estrogen and/or progesterone deficiency to explain the linkbetween KCS and menopause. However, recent research has focused onandrogens, specifically testosterone, and/or metabolized androgens.

It has been shown that in meibomian gland dysfunction, a deficiency inandrogens results in loss of the lipid layer, specificallytriglycerides, cholesterol, monounsaturated essential fatty acids (eg,oleic acid), and polar lipids (eg, phosphatidylethanolamine,sphingomyelin). The loss of polar lipids (present at the aqueous-tearinterface) exacerbates the evaporative tear loss, and the decrease inunsaturated fatty acids raises the melting point of meibum, leading tothicker, more viscous secretions that obstruct ductules and causestagnation of secretions. Patients on anti-androgenic therapy forprostate disease also have increased viscosity of meibum, decreased tearbreak-up time, and increased tear film debris, all indicative of adeficient or abnormal tear film.

It is known that in various tissues pro-inflammatory cytokines may causecellular destruction. For example including interleukin 1 (IL-1),interleukin 6 (IL-6), interleukin 8 (IL-8), TGF-beta, TNF-alpha, andRANTES, are altered in patients with KCS. IL-1 beta and TNF-alpha, whichare present in the tears of patients with KCS, cause the release ofopioids that bind to opioid receptors on neural membranes and inhibitneurotransmitter release through NF-K β production. IL-2 also binds tothe delta opioid receptor and inhibits cAMP production and neuronalfunction. This loss of neuronal function diminishes normal neuronaltone, leading to sensory isolation of the lacrimal gland and eventualatrophy.

Pro-inflammatory neurotransmitters, such as substance P and calcitoningene related peptide (CGRP), are released, which recruit and activatelocal lymphocytes. Substance P also acts via the NF-AT and NF-K βsignaling pathway leading to ICAM-1 and VCAM-1 expression, adhesionsmolecules that promote lymphocyte homing and chemotaxis to sites ofinflammation. Cyclosporin A is an NK-1 and NK-2 receptor inhibitor thatcan down-regulate these signaling molecules and is a novel addition tothe therapeutic armamentarium for dry eye, being used to treat bothaqueous tear deficiency and meibomian gland dysfunction. It has beenshown to improve the goblet cell counts and to reduce the numbers ofinflammatory cells and cytokines in the conjunctiva.

These pro-inflammatory cytokines, in addition to inhibiting neuralfunction, may also convert androgens into estrogens, resulting inmeibomian gland dysfunction, as discussed above. An increased rate ofapoptosis is also seen in conjunctival and lacrimal acinar cells,perhaps due to the cytokine cascade. Elevated levels of tissue-degradingenzymes called matrix metalloproteinases (MMPs) are also present in theepithelial cells.

Mucin synthesizing genes, designated MUC1-MUC17, representing bothtransmembrane and goblet-cell secreted, soluble mucins, have beenisolated, and their role in hydration and stability of the tear film arebeing investigated in patients with dry eye syndrome. Particularlysignificant is MUC5AC, expressed by stratified squamous cells of theconjunctiva and whose product is the predominant component of the mucouslayer of tears. A defect in this and other mucin genes may be a factorin dry eye syndrome development. In addition to dry eye, otherconditions, such as ocular cicatricial pemphigoid, Stevens-Johnsonsyndrome, and vitamin A deficiency, which lead to drying orkeratinization of the ocular epithelium, eventually lead to goblet cellloss. Both classes of mucins are decreased in these diseases, and, on amolecular level, mucin gene expression, translation, andposttranslational processing are altered.

Normal production of tear proteins, such as lysozyme, lactoferrin,lipocalin, and phospholipase A2, is decreased in KCS.

It is clear from the above discussion that common causes of dry eyesyndromes may be ameliorated by treatment with anti-inflammatory agentssuch as topical corticosteroids, topical cyclosporine A and/ortopical/systemic omega-3 fatty acids.

Dry Eye References

Basic teachings regarding dry eye can be found in the followingreferences:

-   Dry Eye Workshop (DEWS) Committee. 2007 Report of the Dry Eye    Workshop (DEWS). Ocul Surf. April 2007; 5(2):65-204.-   Behrens A, Doyle J J, Stern L, et al. Dysfunctional tear syndrome: a    Delphi approach to treatment recommendations. Cornea. September    2006; 25(8):900-7. [Medline].-   Abelson M B. Dry eye, today and tomorrow. Review in Ophthalmology.    2000; 11:132-34.-   American Academy of Ophthalmology. External disease and cornea. In:    Section Seven: Basic & Clinical Science Course. American Academy of    Ophthalmology; 2007-2008.-   Barabino S, Rolando M, Camicione P, et al. Systemic linoleic and    gamma-linolenic acid therapy in dry eye syndrome with an    inflammatory component. Cornea. March 2003; 22(2):97-101. [Medline].-   Bron A J, Tiffany J M, Gouveia S M, et al. Functional aspects of the    tear film lipid layer. Exp Eye Res. March 2004; 78(3):347-60.    [Medline].-   Geerling G, Maclennan S, Hartwig D. Autologous serum eye drops for    ocular surface disorders. Br J Ophthalmol. November 2004;    88(11):1467-74. [Medline].-   Gilbard J P. Dry eye disorders. In: Albert D M, Jakobiec F A, eds.    Principles and Practice of Ophthalmology. Vol 2. WB Saunders Co;    2000:982-1000.-   Karadayi K, Ciftci F, Akin T, et al. Increase in central corneal    thickness in dry and normal eyes with application of artificial    tears: a new diagnostic and follow-up criterion for dry eye.    Ophthalmic Physiol Opt. November 2005; 25(6):485-91. [Medline].-   McCulley J P, Shine W E. The lipid layer of tears: dependent on    meibomian gland function. Exp Eye Res. March 2004; 78(3):361-5.    [Medline].-   Murube J, Nemeth J, Hoh H, et al. The triple classification of dry    eye for practical clinical use. Eur J Ophthalmol. November-December    2005; 15(6):660-7. [Medline].-   Ohashi Y, Dogru M, Tsubota K. Laboratory findings in tear fluid    analysis. Clin Chim Acta. Jul. 15, 2006; 369(1):17-28. [Medline].-   Perry H D, Donnenfeld E D. Dry eye diagnosis and management in 2004.    Curr Opin Ophthalmol. August 2004; 15(4):299-304. [Medline].-   Pflugfelder S C. Advances in the diagnosis and management of    keratoconjunctivitis sicca. Curr Opin Ophthalmol. August 1998;    9(4):50-3. [Medline].-   Stern M E, Gao J, Siemasko K F, et al. The role of the lacrimal    functional unit in the pathophysiology of dry eye. Exp Eye Res.    March 2004; 78(3):409-16. [Medline].-   Tatlipinar S, Akpek E K. Topical ciclosporin in the treatment of    ocular surface disorders. Br J Ophthalmol. October 2005;    89(10):1363-7. [Medline].-   Yoon K C, Heo H, Im S K, et al. Comparison of autologous serum and    umbilical cord serum eye drops for dry eye syndrome. Am J    Ophthalmol. July 2007; 144(1):86-92. [Medline].-   Zoukhri D. Effect of inflammation on lacrimal gland function. Exp    Eye Res. May 2006; 82(5):885-98. [Medline].

Associated AMD References

Associated AMD teachings can be found in the following references:

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Am J Ophthalmol,    1978; 85:28-34.-   6) Smiddy W E, Fine S L. Prognosis of patients with bilateral    macular drusen. Ophthalmol, 1984; 91:271-277.-   7) Friedman D S, O'Colmain B J, Munoz B, et al. (Eye Disease    Prevalence Research Group.) Prevalence of age-related macular    degeneration in the United States. Arch Ophthalmol, 2004;    122:564-572.-   8) Age-Related Eye Disease Study Research Group. A randomized,    placebo-controlled, clinical trial of high-dose supplementation with    vitamins C and E, beta-carotene, and zinc for age-related macular    degeneration and vision loss: AREDS Report No. 8. Arch Ophthalmol,    2001; 119:1417-36.-   9) Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and    Carotenoids. Washington, D.C.: Academy Press; 2000.-   10) Khachik F, Spangler C J, Smith J C, Jr., Canfield L M, Steck A,    Pfander H. Identification, quantification, and relative    concentrations of carotenoids and their metabolites in human milk    and serum. Anal Chem, 1997; 69(10):1873-1881.-   11) Bone R A, Landrum J T, Tarsis S L. Preliminary identification of    the human macular pigment. Vision Res, 1985; 25(11):1531-1535.-   12) Chew E Y, SanGiovanni J P. Lutein. Encyclopedia of Dietary    Supplements, pp. 409-420, Marcel Dekker, Inc., 2005.-   13) SanGiovanni J P, Chew E Y. The role of omega-3 long-chain    polyunsaturated fatty acids in health and disease of the retina.    Progress in Retinal and Eye Research, 2005; 24:87-138.-   14) Neuringer M. in Lipids, Learning, and the Brain: Fats in Infant    Formulas, 103rd Ross Conference on Pediatric Research (ed. Dabbing,    J.), 1993, 134-158 (Ross Laboratories, Adeliade, South Australia).-   15) Fliesler S J, Anderson R E. Chemistry and metabolism of lipids    in the vertebrate retina. Prog Lipid Res, 1983; 22:79-131.-   16) Litman B J, Mitchell D C. A role for phospholipid    polyunsaturation in modulating membrane protein function. Lipids,    1996; 31(Suppl):S193-7.-   17) Litman B J, Niu S L, Polozova A, Mitchell D C. The role of    docosahexaenoic acid containing phospholipids in modulating G    protein-coupled signaling pathways: Visual transduction. J Mol    Neurosci, 2001; 16(2-3):237-242; discussion 279-284.-   18) Schaefer E J, Robins S J, Patton G M, et al. Red blood cell    membrane phosphatidylethanolamine fatty acid content in various    forms of retinitis pigmentosa. J Lipid Res, 1995; 36(7):1427-1433.-   19) Hoffman D R, Birch D G. Docosahexaenoic acid in red blood cells    of patients with X-linked retinitis pigmentosa. Invest Ophthalmol    Vis Sci, 1995; 36(6):1009-1018.-   20) Hoffman D R, Uauy R, Birch D G. Metabolism of omega-3 fatty    acids in patients with autosomal dominant retinitis pigmentosa. Exp    Eye Res, 1995; 60(3):279-289.-   21) Martinez M, Vazquez E, Garcia-Silva M T, et al. Therapeutic    effects of docosahexaenoic acid ethyl ester in patients with    generalized peroxisomal disorders. Am J Clin Nutr, 2000; 71(1    Suppl):376S-385S.-   22) Jumpsen J, M. T. C. Brain Development: Relationship to Dietary    Lipid and Lipid Metabolism. 1997; Champaign, Ill.: AOCS Press.-   23) Clandinin M T, Jumpsen J, Suh M. Relationship between fatty acid    accretion, membrane composition, and biologic functions. J Pediatr,    1994; 125(5 Pt 2):S25-32.-   24) Salem N, Jr., Litman B, Kim H Y, Gawrisch K. Mechanisms of    action of docosahexaenoic acid in the nervous system. Lipids, 2001;    36(9):945-959.-   25) Chen Y, Houghton L A, Brenna J T, Noy N. Docosahexaenoic acid    modulates the interactions of interphotoreceptor retinoid-binding    protein with 11-cis-retinal. J Biol Chem, 1996; 271(34):20507-20515.-   26) de Urquiza, A M et al. Docosahexaenoic acid, a ligand for the    retinoid X receptor in mouse brain. Science, 2000; 290:2140-4.-   27) Lin Q, Ruuska S E, Shaw N S, dong D, Noy N. Ligand selectivity    of the peroxisome proliferator-activated receptor alpha.    Biochemistry, 1999; 38:185-90.-   28) Dreyer C, et al. Positive regulation of the peroxisomal    beta-oxidation pathway by fatty acids through activation of    peroxisome proliferator-activated receptors (PPAR). Biol Cell, 1993;    77:67-76.-   29) Yu K, et al. Differential activation of peroxisome    proliferator-activated receptors by eicosanoids. J Biol Chem, 1995;    270:23975-83.-   30) Politi L E, Rotstein N P, Carri N G. Effect of GDNF on    neuroblast proliferation and photoreceptor survival: additive    protection with docosahexaenoic acid. Invest Ophthalmol Vis Sci,    2001; 42(12):3008-3015.-   31) Rotstein N P, Aveldano M I, Barrantes F J, Roccamo A M, Politi    L E. Apoptosis of retinal photoreceptors during development in    vitro: protective effect of docosahexaenoic acid. J Neurochem, 1997;    69(2):504-513.-   32) Rotstein N P, Politi L E, Aveldano M I. Docosahexaenoic acid    promotes differentiation of developing photoreceptors in culture.    Invest Ophthalmol Vis Sci, 1998; 39(13):2750-2758.-   33) Rotstein N P, Aveldano M I, Barrantes F J, Politi L E.    Docosahexaenoic acid is required for the survival of rat retinal    photoreceptors in vitro. J Neurochem, 1996; 66(5):1851-1859.-   34) Kim H Y, Akbar M, Kim K Y. Inhibition of neuronal apoptosis by    polyunsaturated fatty acids. J Mol Neurosci, 2001; 16(2-3):223-227;    discussion 279-284.-   35) Diep Q N, Amiri F, Youyz R M, et al. PPARalpha activator effects    on Ang II-induced vascular oxidative stress and inflammation.    Hypertension, 2002; 40(6):866-871.-   36) Yang S P, Morita I, Murota S I. Eicosapentaenoic acid attenuates    vascular endothelial growth factor-induced proliferation via    inhibiting Flk-1 receptor expression in bovine carotid artery    endothelial cells. J Cell Physiol, 1998; 176(2):342-349.-   37) von Knethen A, Callsen D, Brune B. Superoxide attenuates    macrophage apoptosis by NF-kappa B and AP-1 activation that promotes    cyclooxygenase-2 expression. J Immunol, 1999; 163(5):2858-2866.-   38) Morita I, Zhang Y W, Murata S I. Eicosapentaenoic acid protects    endothelial cell function injured by hypoxia/reoxygenation. Ann N Y    Aced Sci, 2001; 947:394-397.-   39) Calder P C. Polyunsaturated fatty acids, inflammation, and    immunity. Lipids, 2001; 36(9): 1007-1024.-   40) Rose D P, Connolly J M, Rayburn J, Coleman M. Influence of diets    containing eicosapentaenoic or docosahexaenoic acid on growth and    metastasis of breast cancer cells in nude mice. J Natl Cancer Inst,    1995; 87(8):587-592.-   41) Rose D P, Connolly J M. Antiangiogenicity of docosahexaenoic    acid and its role in the suppression of breast cancer cell growth in    nude mice. Int J Oncol, 1999; 15(5):1011-1015.-   42) Badawi A F, El-Sohemy A, Stephen L L, Ghoshal A K, Archer M C.    The effect of dietary n-3 and n-6 polyunsaturated fatty acids on the    expression of cyclooxygenase 1 and 2 and levels of p21as in rat    mammary glands. Carcinogenesis, 1998; 19(5):905-910.-   43) Hamid R, Singh J, Reddy B S, Cohen L A. Inhibition of dietary    menhaden oil of cyclooxygenase-1 and -2 in    N-nitrosomethylurea-induced rat mammary tumors. Int J Oncol, 1999;    14(3):523-528.-   44) Ringbom T, Huss U, Stenholm A, et al. Cox-2 inhibitory effects    of naturally occurring and modified fatty acids. J Nat Prod, 2001;    64(6):745-749.-   45) Kanayasu T, Morita I, Nakao-Hayashi J, et al. Eicosapentaenoic    acid inhibits tube formation of vascular endothelial cells in vitro.    Lipids, 1991; 26(4):271-276.-   46) Farrara N, Davis-Smyth T. The biology of vascular endothelial    growth factor. Endocr Rev, 1997; 18(1):4-25.-   47) Mares-Perlman J A, Brady W E, Klein R, VandenLangenberg G M,    Klein B E, Plata M. Dietary fat and age-related maculopathy. Arch    Ophthalmol, 1995; 113(6):743-8.-   48) Heuberger R A, Mares-Perlman J A, Klein R, Klein B E, Millen A    E, Palta M. Relationship of dietary fat to age-related maculopathy    in the Third National Health and Nutrition Examination Survey. Arch    Ophthalmol, 2001; 119(12):1833-8.-   49) Smith W, Mitchell P, Leeder S R. Dietary fat and fish intake and    age-related maculopathy. Arch Ophthalmol, 2000; 118(3):401-4.-   50) Seddon J M, Rosner B, Sperduto R D, Yannuzzi L, Haller J A,    Blair N P, Willett W. Dietary fat and risk for advanced age-related    macular degeneration. Arch Ophthalmol, 2001; 119(8):1191-9.-   51) Seddon J M, Cote J, Rosner B. Progression of age-related macular    degeneration: Association with dietary fat, transunsaturated fat,    nuts, and fish intake. Arch Ophthalmol, 2003; 121(12):1728-1737.-   52) SanGiovanni J P, Chew E Y, Clemons T E, Seddon J M, Klein R,    Age-Related Eye Disease Study (AREDS) Research Group. Dietary lipids    intake and incident advanced Age-Related Macular Degeneration (AMD)    in the Age-Related Eye Disease Study (AREDS). Annual Meeting, May    2005, Association for Research in Vision and Ophthalmology (ARVO),    Fort Lauderdale, Fla.-   53) Seddon J M, Ajani D A, Sperduto R D, et al. Dietary carotenoids,    vitamins A, C and E, and advanced age-related macular degeneration.    Eye Disease Case-Control Study Group. JAMA, 1994; 272(18):1413-20.-   54) Snellen E L, Verbeek A L, Van Den Hoogen G W, Cruysberg J R,    Hoyng C B. Neovascular age-related macular degeneration and its    relationship to antioxidant intake. Acta Ophthalmol Scand, 2002;    80(4):368-71.-   55) Mares-Perlman J A, Fisher A I, Klein R, et al. 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Five of six studies examining the association of dietarylutein/zeaxanthin intake with advanced AMD have yielded inverserelationships that are statistically significant. The magnitude of oddsratios in these studies ranged from 0.1 to 0.7. Both sets of findingsare germane in guiding applied clinical research on prevention andtreatment of retinal disease, since: (1) tissue concentrations of DHA,lutein, and zeaxanthin per unit area are substantially higher in theretina than elsewhere in the body; and (2) retinal tissue status ofthese compounds is modifiable and dependent upon intake.

The AREDS II study protocol (concluded its scientific rational bystating: “There is a compelling need to implement a clinical trial onnutrients that are both concentrated in the retina and implicated inmodulation of pathogenic factors and processes of AMD.”

It has been well established that lutein and trans-zeaxanthin arepresent in human retinal tissue and that they function to protect theeye from photo induced injury. The CARMIS study, which included amixture of lutein, trans-zeaxanthin and astaxanthin, is the onlyclinical trial which reported the use of astaxanthin. Unfortunately,there have been no reports of the use of astaxanthin alone in any humanclinical trial for the prevention or amelioration of dry AMD. The CARMISstudy failed to determine if supplementation with astaxanthin alone is akey determinate of the positive outcomes of the study or thatastaxanthin deposited on retinal epithelial cells. One possibleinterpretation of the CARMIS study is that lutein and zeaxanthin aloneprovided the observed benefits of the formulation employed, or inanother interpretation that astaxanthin in combination with lutein andzeaxanthin provided the observed benefits. However, in no possibleinterpretation can one conclude unequivocally that astaxanthin aloneprevents or ameliorates dry AMD.

In addition, the work of Tso, though claiming utility of astaxanthin forprevention or amelioration of dry AMD in humans, was not based onclinical trials performed on human subjects but instead on a differentmammalian species, namely in rats.

Therefore, there remains no conclusive evidence that astaxanthin alonecan prevent or ameliorate dry AMD in man since no human study has everbeen performed using astaxanthin supplementation alone, nor has anyhuman study shown that astaxanthin actually deposits anywhere in thehuman retina, the first required step to retinal protection by thispowerful carotenoid.

Potential Roles of Polyunsaturated Fatty Acids in Eye Physiology

An inverse relationship of dietary omega-3 LCPUFA intake with advancedAMD has been reported in six studies examining the issue. For prevalentdisease, the magnitude of odds ratios for highest versus lowest omega-3LCPUFA intake ranged from 0.4 to 0.9.

Among these studies, the one containing the largest number of subjectswith neovascular or “wet” AMD yielded a significantly lower likelihoodof having the disease among participants reporting the highestconsumption of omega-3.

The scientific literature is replete with the certain human benefits oftriacylglyceride bound EPA and DHA found in fish oil and fish oilconcentrates and more recently the potential utility of phospholipidbound EPA and DHA found in krill oil derived from Euphasia superba orAntarctic krill.

The cardiovascular benefits as well as the anti-inflammatory benefits ofsuch fish and krill oils⁶⁶⁻⁶⁷, and in particular triacylglyceride boundEPA and DHA derived from fish oils as well as algae derivedtriacylglyceride bound DHA are well known⁶⁸⁻⁷³. Such algae derived DHAis used in large part as a supplement in infant formulas to ensure brainhealth in the developing fetus and in infants.

LCPUFAs affect factors and processes implicated in the pathogenesis ofvascular and neural retinal disease.¹³ Evidence characterizingstructural and functional properties of LCPUFAs indicates that thesenutrients may operate both as: (1) essential factors in thevisual-sensory process, and (2) protective agents against retinaldisease.

Docosahexaenoic Acid (DHA) is the major structural lipid of retinalphotoreceptor outer segment membranes.¹⁴⁻¹⁵ Tissue DHA status affectsretinal cell signaling mechanisms involved in phototransduction.¹⁶⁻¹⁷Tissue DHA insufficiency is associated with conditions characterized byalterations in retinal function,¹⁸⁻²⁰ and functional deficits have beenameliorated with DHA supplementation in some cases.²¹ Biophysical andbiochemical properties of DHA may affect photoreceptor function byaltering membrane permeability, fluidity, thickness, and lipid phaseproperties.²²⁻²³ DHA may operate in signaling cascades to enhanceactivation of membrane-bound retinal proteins.^(16-17,24) DHA may alsobe involved in rhodopsin regeneration.²⁵

DHA and Eicosapentaenoic Acid (EPA) may serve as protective agentsbecause of their effect on gene expression,²⁶⁻²⁹ retinal celldifferentiation,³⁰⁻³² and survival.³⁰⁻³⁴ DHA activates a number ofnuclear hormone receptors that operate as transcription factors formolecules that modulate redox-sensitive and proinflammatory genes; theseinclude the peroxisome proliferator-activated receptor-α (PPAR-α)²⁷ andthe retinoid X receptor (RXR).²⁶ In the case of PPAR-α, this action isthought to prevent endothelial cell dysfunction and vascular remodelingthrough inhibition of vascular smooth muscle cell proliferation,inducible nitric oxide synthase production, interleukin (IL)-1 inducedcyclooxygenase (COX)-2 production, and thrombin-induced endothelin-1production.³⁵

Research on model systems demonstrates that omega-3 LCPUFAs also havethe capacity to affect production and activation of angiogenic growthfactors,³⁶⁻³⁸ arachidonic acid-based proangiogenic eicosanoids,³⁹⁻⁴³ andmatrix metalloproteinases involved in vascular remodeling.⁴⁴

EPA depresses vascular endothelial growth factor (VEGF)-specifictyrosine kinase receptor activation and expression.^(36,45) VEGF playsan essential role in induction of endothelial cell migration andproliferation, microvascular permeability, endothelial cell release ofmetalloproteinases and interstitial collagenases, and endothelial celltube formation.⁴⁶ The mechanism of VEGF receptor down-regulation isbelieved to occur at the tyrosine kinase nuclear factor-kappa B (NFkB)site because EPA treatment causes suppression of NFkB activation. NFkBis a nuclear transcription factor that up-regulates COX-2 expression,intracellular adhesion molecule (ICAM), thrombin, and nitric oxidesynthase. All four factors are associated with vascular instability.³⁵COX-2 drives conversion of arachidonic acid to a number of angiogenicand pro-inflammatory eicosanoids.

Although the mechanistic benefits of dietary supplementation with EPAand DHA polyunstaruated fatty acids in triacylglyceride form are wellknow, it remains speculative that such triacylglyceride bound EPA andDHA can improve vision. Such hypothesis is now under exploration underthe National Eye Institute's 5-year AREDS II study.

Krill Oil

Nowhere does the literature teach that phospholipid bound EPA and DHAderived from Antarctic krill imparts any benefit in ameliorating eyerelated diseases such as AMD and/or syndromes such as dry eye syndrome,although more recent research indicates that krill oil extractscontaining some phospholipid bound EPA and DHA may be useful in thetreatment of hyperlipidemia, joint disease as manifested inosteoarthritis and/or rheumatoid arthritis, blood sugar control andattention deficit hyperactivity disorder.⁷⁴

However one must use caution when evaluating such information since allkrill oil clinical trials to date have been conducted using krill oilthat contains a mixture of triacylglyceride bound and phospholipid boundEPA and DHA. In addition, such krill oils usually contain approximately30-40% weight-weight phospholipid bound fatty acids, principally in theform of saturated phosphatidylcholines which themselves are importantcellular membrane components.

Thus, it is difficult at the present time, to distinguish which form ofEPA and DHA present in krill oil is useful as reported in the referencescited above as well as the clinical trials described therein. It is alsowell known that phospholipids in general act as excellent emulsifiersand are known to improve the stability of emulsions and thebio-availability of many active ingredients. Phospholipids also play animportant role in the production of micelle based drug delivery systemscontaining active ingredients with vastly improved bio-availability.Therefore it remains undetermined what the clinical value of krill oil,either alone or in combination with carotenoids, is in the prevention oramelioration of eye related diseases such as AMD, cataracts or dry eyesyndromes.

Cataracts

A cataract is an opacity, or clouding, of the lens of the eye. Theprevalence of cataracts increases dramatically with age. It typicallyoccurs in the following way. The lens is an elliptical structure thatsits behind the pupil and is normally transparent. The function of thelens is to focus light rays into images on the retina (thelight-sensitive tissue at the back of the eye).

In young people, the lens is elastic and changes shape easily, allowingthe eyes to focus clearly on both near and distant objects. As peoplereach their mid-40s, biochemical changes occur in the proteins withinthe lens, causing them to harden and lose elasticity. This causes anumber of vision problems. For example, loss of elasticity causespresbyopia, or far-sightedness, requiring reading glasses in almosteveryone as they age.

In some people, the proteins in the lens, notably those called alphacrystallins, may also clump together, forming cloudy (opaque) areascalled cataracts. They usually develop slowly over several years and arerelated to aging. In some cases, depending on the cause of thecataracts, loss of vision progresses rapidly. Depending on how densethey are and where they are located, cataracts can block the passage oflight through the lens and interfere with the formation of images on theretina, causing vision to become cloudy.

Nuclear cataracts form in the nucleus (the inner core) of the lens. Thisis the most common variety of cataract associated with the agingprocess. Cortical cataracts form in the cortex (the outer section of thelens). Posterior subcapsular cataracts form toward the back of acellophane-like capsule that surrounds the lens. They are more frequentin people with diabetes, who are overweight, or those taking steroids.Although the causes of cataract formation remain largely unknown,researchers have been focusing on particles called oxygen-free radicalsas a major factor in the development of cataracts. They cause harm inthe following way:

Oxygen-free radicals (also called oxidants) are molecules produced bynatural chemical processes in the body. Toxins, smoking, ultravioletradiation, infections, and many other factors can create reactions thatproduce excessive amounts of these oxygen-free radicals. When oxidantsare overproduced, these chemical reactions can be very harmful to nearlyany type of cell in the body. At times these reactions can even affectgenetic material in cells.

Cataract formation is one of many destructive changes that can occurwith overproduction of oxidants, possibly in concert with deficienciesof an important protective anti-oxidant called glutathione. Glutathioneoccurs in high levels in the eye and helps clean up these free radicals.One theory is that in the aging eye, barriers develop that preventglutathione and other protective antioxidants from reaching the nucleusin the lens, thus making it vulnerable to oxidation. Sunlight consistsof ultraviolet (referred to as UVA or UVB) radiation, which penetratesthe layers of the skin. Both UVA and UVB have destructive propertiesthat can promote cataracts. The eyes are protected from the sun byeyelids and the structure of the face (overhanging brows, prominentcheekbones, and the nose). Long-term exposure to sunlight, however, canovercome these defenses.

UVB radiation produces the shorter wavelength, and primarily affects theouter skin layers. It is the primary cause of sunburn. It is also the UVradiation primarily responsible for cataracts. Long-term exposure toeven low levels of UVB radiation can eventually cause changes in thelens, including pigment changes, which contribute to cataractdevelopment. (UVB also appears to play a role in macular degeneration,an age-related disorder of the retina.) UVA radiation is composed oflonger wavelengths. They penetrate more deeply and efficiently into theinner skin layers and are responsible for tanning. The main damagingeffect of UVA appears to be the promotion of the release of oxidants.Cataracts are common side effects of total body radiation treatments,which are administered for certain cancers. This observation indicatesthat ionizing radiation, which produces large numbers of free radicalsdramatically accelerates cataract formation.

Glaucoma and its treatments, including certain drugs (notably miotics)and filtering surgery, pose a high risk for cataracts. The glaucomadrugs posing a particular risk for cataracts including demecarium(Humorsol), isoflurophate (Floropryl), and echothiophate (Phospholine).Uveitis is chronic inflammation in the eye, which is often caused by anautoimmune disease or response. Often the cause is unknown. It is a rarecondition that carries a high risk for cataracts. It is not clearwhether nutrition plays a significant role in cataract development. Darkcolored (green, red, purple, and yellow) fruits and vegetables usuallyhave high levels of important plant chemicals (phytochemicals) and maybe associated with a lower risk for cataracts.

In analyzing nutrients, researchers have focused on antioxidants andcarotenids. Studies have not demonstrated that antioxidant vitaminsupplements (such as vitamins C and E) help prevent cataracts. Luteinand zeaxanthin are the two carotenids that have been most studied forcataract prevention. They are xanthophylis compounds, which are aparticular type of carotenid. Lutein and zeaxanthin are found in thelenses of the eyes. Some evidence indicates that xanthophyll-rich foods(such as dark green leafy vegetables) may help retard the aging processin the eye and protect against cataracts. However, there is not enoughevidence to suggest that taking supplements with these carotenoidslowers the risk of cataract formation. Since little is known about theexact mechanism for formation of cataracts, it is not surprising thatthere are no known drugs or dietary supplements including thecarotenoids that prevent cataract formation there remains a need to finda suitable preventative treatment to prevent or ameliorate furthercataract formation. Since no drugs can reverse nor prevent cataractformation, the only current treatment suitable for advanced cataract inhumans is lens replacement surgery.

Cataract References

Basic teachings regarding cataracts can be found in the followingreferences:

Allen D. Cataract. BMJ Clinical Evidence. Web publication date: 1 Apr.2007 (based on October 2006 search). Accessed Jul. 1, 2008.

American Academy of Ophthalmology. Cataract in the Adult Eye, PreferredPractice Pattern. San Francisco: American Academy of Ophthalmology,2006. Accessed Jul. 1, 2008.

Awasthi N, Guo S, Wagner B J. Posterior capsular opacification: aproblem reduced but not yet eradicated. Arch Ophthalmol. 2009 April;127(4):555-62.

Bell C M, Hatch W V, Fischer H D, Cernat G, Paterson J M, Gruneir A, etal. Association between tamsulosin and serious ophthalmic adverse eventsin older men following cataract surgery. JAMA. 2009 May 20;301(19):1991-6

Clinical Trial of Nutritional Supplements and Age-Related Cataract StudyGroup, Maraini G, Sperduto R D, Ferris F. Clemons T E, Rosmini F, et al.A randomized, double-masked, placebo-controlled clinical trial ofmultivitamin supplementation for age-related lens opacities. Clinicaltrial of nutritional supplements and age-related cataract report no. 3.Ophthalmology. 2008 April; 115(4):599-607.e1.

Fernandez M M, Afshari N A. Nutrition and the prevention of cataracts.Curr Opin Ophthalmol. 2008 January; 19(1):66-70.

Friedman A H. Tamsulosin and the intraoperative floppy iris syndrome.JAMA. 2009 May 20; 301(19):2044-5.

Guercio J R, Martyn L J. Congenital malformations of the eye and orbit.Otolaryngol Clin North Am. 2007 February; 40(1):113-40, vii.

Long V, Chen S, Hatt S. Surgical interventions for bilateral congenitalcataract. Cochrane Database Syst Rev. 2006 Jul. 19; 3:CD003171.

Moeller S M, Voland R, Tinker L, Blodi B A, Klein M L, Gehrs K M, et al.Associations between age-related nuclear cataract and lutein andzeaxanthin in the diet and serum in the Carotenoids in the Age-RelatedEye Disease Study, an Ancillary Study of the Women's Health Initiative.Arch Ophthalmol. 2008 March; 126(3):354-64.

Olitsky S E, Hug D, and Smith L P. Abnormalities of the lens. In:Kliegman R M, Behrman R E, Jenson H B, Stanton B F, eds. Nelson Textbookof Pediatrics. 18th ed. St. Louis, Mo.: WB Saunders; 2007; chap 627.

Wishart M S, Dagres E. Seven-year follow-up of combined cataractextraction and viscocanalostomy. J Cataract Refract Surg. 2006 December;32(12):2043-9.

The ability of a carotenoid to pass the blood-retinal brain barrier isimportant because carotenoids are not synthesized by the human body. Theonly source of carotenoids for humans is dietary intake. Furthermore,humans have a very limited ability to modify carotenoids. Therefore, thecarotenoids accumulate in various organs in the ingested form.Accordingly, if a particular carotenoid is unable to cross theblood-retinal brain barrier, the carotenoid cannot accumulate in theretina and serve as an antioxidant.

Furthermore, some carotenoids that are not normal constituents of humanplasma, but have the ability to cross the blood-retinal brain barrier,have demonstrated adverse affects on the retina. Canthaxanthin which isintentionally ingested to provide an artificial suntan has accumulatedin the retina in the form of crystals and has temporarily affected eyeadaptation to the dark. In addition, beta-carotene has a very limitedability to cross the blood-retinal brain barrier.

Therefore, even though the carotenoids are known as strong antioxidantsand are present in abundant supply, the carotenoids have not been usedfor the treatment of central nervous system damage, or eye damage,caused by disease or injury. The carotenoids investigated to date eithercould not effectively cross the blood-retinal barrier (i.e.,beta-carotene) or adversely affected the eye (i.e., canthaxanthin).

In accordance with an important feature, the composition comprises atherapeutically effective amount of a synergistic multi-ingredientcomposition of mixed carotenoids including at least S,S′-astaxanthinderived from Haematococcus pluvialis, and one or more of lutein and/ortrans-zeaxanthin or meso-zeaxanthin admixed with a therapeuticallyeffective amount of krill oil containing phospholipid bound andtriglyceride bound EPA and DHA. The composition includes 50 to 500 mg ofkrill oil, 0.5 to 8 mg of astaxanthin, 2 to 15 mg of lutein and 0.2 to12 mg of trans-zeaxanthin. The composition contains allnaturally-occurring compounds and is a potent antioxidant andanti-inflammatory composition, which can be is used in a method toameliorate and retard, or prevent, cell damage in an individualsuffering from a degenerative, inflammatory disease or injury to theeye. In accordance with another important feature, the administration ofa therapeutically-effective amount of the composition to an individualprevents, retards and/or ameliorates free radical-induced damageresulting from eye disease or injury. For example, damage to a retinacan result from either photic injury, neurodegenerative disease or anischemic insult followed by reperfusion. With respect to damage fromphotic injury, the composition decreases the loss of photoreceptorcells. With respect to damage from ischemic insult, the compositionameliorates the loss of ganglion cells and the inner layers of theretinal neuronal network.

Interestingly, none of the carotenes tested to date, and most of thexanthophylls tested to date do not pass through the blood brain barrierwith a few notable exceptions. These exceptions include lutein,trans-zeaxanthin, canthaxanthin and astaxanthin.

Human serum typically contains about ten carotenoids. The majorcarotenoids in human serum include beta-carotene, alpha-carotene,cryptoxanthin, lycopene and lutein. Small amounts of zeaxanthin,phytofluene and phytoene are also found in human organs. However, of allof these carotenoids, only zeaxanthin and lutein are found in the humanretina. In addition to certain carotenoids, the retina also has thehighest concentration of polyunsaturated fatty acids of any tissue inthe human body. These polyunsaturated fatty acids are highly susceptibleto free radial and singlet oxygen induced decomposition. Therefore thereis an absolute need to protect these polyunsaturated fatty acids, whichmake up a portion of the cellular membrane bi-layer, from photo inducedfree radical or singlet oxygen degradation.

It has been theorized that zeaxanthin and lutein are concentrated in theretina because of their ability to quench singlet oxygen and to scavengefree radicals, because they pass the blood and eye brain barriers andare required in the oxygen rich environment of the retina to preventlight mediated free radical damage to the retina.

In fact, zeaxanthin is the predominant carotenoid found in the centralportion of the retina and more specifically is located in concentrationin the retinal cones located in the central area of the retina (ie. themacula). Lutein, on the other hand, is located in the peripheral area ofthe retina in the rod cells. Therefore, the eye preferentiallyaccumulates zeaxanthin over lutein in the critical central macularretinal area, (zeaxanthin interestingly, is a much more effectivesinglet oxygen scavenger than lutein), where the greatest level of lightimpinges.

Biochemists have determined the exact, yet complicated, mechanism forlight sensory response in the eye. It involves a key protein calledrhodopsin whose structure includes a bound polyunsaturated compoundcalled retinal (retinal is structurally related to vitamin A). Whenlight enters the eye, cis-retinal isomerizes to all its all-transisomer, causing disassociation of itself from its protein carrier. Thedisassociation triggers a complicated cascade leading to nerve basedtransmission of electrons to the brain via the optic nerve. All of this“photochemistry” takes a mere 200 femtoseconds to occur making it one ofthe fastest biochemical to electron transformations known.

Chemists have learned that the retina is highly susceptible topolymerization by localized free radicals and highly reactive singletoxygen. Because the retina is a strong absorber of light and because theretina is highly vascularized and thus rich in dissolved oxygen, naturehas provided zeaxanthin as the key retinal carotenoid for protection ofthe central foveal region of the retina from light induced damage atthat point in the center of the retina where the most significant lightimpingement occurs.

Clinical studies in man indicate that photic injury is a cause of agerelated macular degeneration because of the cumulative effect ofrepeated photic insult leading to the gradual loss of photoreceptorcells.

There have been many clinical trials designed to support thesupplementation of the diet with lutein, however, as of 2007, thereappears to be no unequivocal evidence that lutein supplementation isnecessary in eye healthcare despite its wide acceptance as a supplement.This may simply imply that supplementation with extra lutein is notnecessary since it is a readily available xanthophyll in manyvegetables. More recently trans-zeaxanthin and meso-zeaxanthin have alsoentered the marketplace as an eye healthcare supplements which indeedmakes sense. However, is there yet a better carotenoid meeting all therequirements associated with eye/blood/brain barrier transport,accumulation in the macula and capable of long term use? The answer isfound in the xanthophyll astaxanthin.

Dr. Mark Tso, at the Univ. of Ill, has demonstrated that astaxanthin isone such naturally occurring antioxidant meeting all of these criticalcriteria in rats. Astaxanthin is the carotenoid xanthophyll responsiblefor the red color in salmon, lobster, krill, crab, other shell fish andin the micro algae Haematoccous pluvialis. The latter source has madeastaxanthin readily available worldwide for such uses. U.S. Pat. No.5,527,533 was issued to the Univ. of Ill. describing the use ofastaxanthin more fully in eye related diseases and which is herebyincorporated by in its entirety.

In addition, astaxanthin is a much more powerful antioxidant thancanthoaxanthin, beta-carotene, zeaxanthin, lutein and alpha-tocopherol.Shimidzu et al. discovered that astaxanthin is 550 times more potentthan alpha-tocopherol, 27.5 times more potent than lutein and 11 timesmore potent that beta-carotene in quenching singlet oxygen. In addition,Bagchi discovered that natural astaxanthin is 14 times more potent thanalpha-tocopherol, 54 times more potent that beta-carotene and 65 timesmore potent that ascorbic acid (Vitamin C) in scavenging oxygen freeradicals. Thus, though there are dramatic differences in the potency ofastaxanthin when comparing the quenching of singlet oxygen and thescavenging of oxygen free radicals, it is clear that astaxanthincompares very favorably to zeaxanthin and lutein, the two carotenoidsthat are found naturally in the retina.

There is one more aspect of carotenoids, namely that some carotenoidscan act as pro-oxidants. This is important since a carotenoid withpro-oxidant capability actually causes oxidation to occur in the bodywhen high concentrations are present in tissue. Martin, et al. showedthat beta-carotene, lycopene and zeaxanthin can become pro-oxidantsunder certain conditions, however because astaxanthin is the most potentof all carotenoids, Beutner et al. showed that astaxanthin can never benor has it ever exhibited any pro-oxidant activity unlike the zeaxanthinfound in the human eye. Since humans already have an abundant source oflutein and trans-zeaxanthin in their diets from many vegetable sourcesand are already present in the human eye, it appears that astaxanthinwith its unique qualifying properties, unlike lutein ortrans-zeaxanthin, may be the eye healthcare supplement of choice. Withastaxanthin's extraordinarily potent antioxidant properties, its abilityto cross the blood brain/eye barrier and concentrate in the retinalmacula in other mammalian species, without the side effects seen withcanthaxanthin, and in light of Tso's contributions, astaxanthin, in aconvenient dietary supplement presentation, may emerge as thepre-eminent new ingredient addition to lutein and/or zeaxanthin eyehealthcare supplementation for the management of eye related oxidativestress and thus the prevention and mitigation of degenerative diseasesof the eye such as age related macular degeneration (ARMD) and cataractformation if astaxanthin deposition can be experimentally confirmed inhuman retinal tissue.

In addition, Tso found that light induced damage, photo-receptor celldamage, ganglion cell damage and damage to neurons of the inner retinallayers can be prevented or ameliorated by the use of astaxanthinincluding neuronal damage from ischemic, photic, inflammatory anddegenerative insult in rats. Tso's patent claims the use of astaxanthinacross a wide range of eye diseases including age related maculardegeneration, diabetic neuropathy, cystoid macular edema, centralretinal arterial and veneous occlusion, glaucoma and inflammatory eyediseases such as retinitis, uveitis, iritis, keratitis and scleritis,all disease states common to eye insult by oxidative species such asfree radicals however this work was never confirmed in humans.

Oral administration of astaxanthin confirms that it is at leasttransported into human blood stream, however, its deposition in humanretinal tissue has never been confirmed.

Astaxanthin is the major pigment of certain micro algae and crustaceans.Astaxanthin is a lipid-soluble pigment primarily used for pigmentingcultured fish, like salmon, which must ingest astaxanthin to yieldconsumer-acceptable pink-colored salmon muscle. Astaxanthin also is anantioxidant which is about 100 to about 1000 times more effective thanalpha-tocopherol.

The prime source of commercial S,S′-astaxanthin is micro algae, and, toa very small extent, is found in krill oil derived from Euphasia superba(Antarctic Krill). Astaxanthin also is available synthetically, howeversynthetic astaxanthin may not be safe for use in humans since itcontains 3 known enantiomers including R,R′, R, S′ and S,S′ which arenot easily nor economically separated two of which have unknown humansafety data. The preferred naturally-occurring S,S′-astaxanthin can beused in the composition and method of the present invention.

As previously stated, the retinal pigment epithelium protects the retinaby providing a blood-retinal brain barrier. The barrier excludes plasmaconstituents that are potentially harmful to the retina. As alsopreviously stated, the blood-retinal brain barrier only permits luteinand zeaxanthin to enter the retina, and excludes other carotenoidspresent in human serum, including beta-carotene which is the mostabundant carotenoid in human serum. Astaxanthin is not anaturally-occurring constituent in the retina. Therefore, the presenceof a physiologically significant amount of astaxanthin in the retina ofrats may illustrate the ability of astaxanthin to readily cross theblood-retinal brain barrier into the retina of humans. The optimal doseof the composition can be determined by a person skilled in the artafter considering factors such as the disease or injury to be treated,the severity of the central nervous system damage by oraladministration. The daily dose of composition can be administered dailyor in accordance with a regimen determined by a person skilled in theart, with the length of treatment depending upon the severity and natureof the injury to the central nervous system, the need to improveaccommodation or to control dry eye syndrome.

The composition can be administered to an individual orally. Whenadministered orally, the composition, for example, can be in the form ofa liquid preparation, The administration of the composition to anindividual suffering from an eye injury or disease, such as freeradical-induced injury, benefits the vision of the individual bypreventing further photoreceptor cells from damage or destruction. Thefree radical-induced damage can be attributed to light-induced injury orto injury resulting from an ischemic insult and subsequent reperfusionor neurodegenerative diseases. The administration of astaxanthin alsohelps prevent and retard photic injury in addition to amelioratingphotic injury.

The administration of the composition ameliorates photoreceptor celldamage that is light induced, and ameliorates ganglion cell damage thatis induced by ischemic insult and subsequent reperfusion. Theadministration of astaxanthin also retards the progress of degenerativeeye diseases and benefits the vision of individuals suffering from adegenerative eye disease, such as age-related macular degeneration.

The administration of the composition also provides a method of treatingischemic retinal diseases, such as diabetic retinopathy, cystoid macularedema, central retinal arterial occlusion, central retinal venousocclusion and glaucoma. In addition, the composition is useful intreating inflammatory diseases of the eye such as retinitis, uveitis,iritis, keratitis and scleritis wherein free radicals are produced inabundance, the prevention of cataracts and the treatment of certaincauses of dry eye syndromes.

Therefore, the antioxidant properties of the composition, coupled withthe ability of the composition to cross the blood-retinal brain barrier,admixed with anti-inflammatory sources of EPA and DHA and the lack oftoxicity of the composition and the lack of adverse side effectsassociated with the composition, make the composition a usefulcomposition to prevent or ameliorate such eye related diseases, dry eyesyndrome and/or cataracts and dry eye syndromes.

“Reacted” Carotenoids and Delivery System

The analytical analysis of the product blend of krill oil andcarotenoids, including astaxanthin during testing, gave conflictingrecovery data for the added astaxanthin when evaluated using HPLC(High-Performance Liquid Chromatography, also known as High-PressureLiquid Chromatography) analysis. It was determined that over time,astaxanthin apparently, and rapidly, chemically combined in what appearsto be an unknown way with one or more krill oil components to produce acompound that is not detectable using the HPLC methodology used toquantify the amount of astaxanthin in such initially blended product. Asa result, a “capsule in a capsule” was designed to prevent such loss ofastaxanthin to the krill oil in order to retain label claims. This is anunexpected observation/result with great benefits. Further, it isbelieved that astaxanthin when mixed with pure fish oil containingessentially all triacylglycerides bound fatty acids, does not result inquantitative loss of astaxanthin to fish oil components as observed inkrill oils. One skilled in the art would not have anticipated theinstability of astaxanthin and other carotenoids in such added mixtures.

Testing has determined that astaxanthin added to krill oil results in anunstable product with respect to recoverable astaxanthin by HPLCanalysis. A chemical reaction most likely takes place between krill oiland at least astaxanthin in such mixtures. Nevertheless, the astaxanthindoes not seem to be destroyed in such mixtures since UV based analysisindicates that the astaxanthin moiety is still present withoutstructural loss because it is still able to absorb UV at the appropriatequantifying absorption wavelength of astaxanthin at essentially 100%quantification of the amount of astaxanthin added to such blendedproduct. Had UV alone been used to determine product stability thedecrease in recoverable astaxanthin over time would not have beenobserved. The test results, tables and charts of FIGS. 2-6 show thedecrease in recoverable astaxanthin over time.

The carotenoids admixed with krill oil should be able to address notonly the eye diseases known to be ameliorated by carotenoids but alsothe inflammatory diseases of the eye (including but not limited to dryeye syndrome) associated with the known anti-inflammatory activity ofomega-3's. Just because phospholipid bound EPA and DHA are active aloneas an anti-hyperlipidemic or as a reducer of pain in arthritic jointsdoes not insure that such phospholipids from any source such as krilloil would prove effective for the treatment of inflammatory conditionsof the eye. The AREDS2 study cited above is an example. This study wasdesigned to determine if triglyceride bound omega-3 fatty acids would beeffective in amelioration of inflammatory eye diseases.

As noted before, the astaxanthin disappears and is converted to a newproduct in the presence of the krill oil. Other carotenoids can possiblyhave a similar effect. This is an unexpected result such that theaddition of some carotenoids and primarily astaxanthin to krill oilresults in the chemical reaction. The design of the capsule in a capsuleprevents initial mixing.

In accordance with a non-limiting example, FIG. 1 illustrates a medicineor drug delivery system at 10 that includes a central capsule 12containing carotenoids 13 while an outer capsule 14 contains krill oil15 and contains the inner capsule as illustrated. This capsule withinthe capsule prevents the disappearance of the carotenoids and theinability of standard HPLC (high-performance liquid chromatography)methods to quantitatively recover the carotenoids, especially withastaxanthin. Both capsules 12,14 could be formed of gelatin or similarmaterial and formed to prevent mixing of carotenoids and krill oil untilafter capsule ingestion.

When added alone or in combination with other carotenoids, astaxanthinHPLC based recovery quickly abates much to the surprise of those skilledin the art. There is possible trans-esterification occurring betweenkrill oil fatty acid esters and partially esterified carotenoids thatcreates a new compound. It is possible that the compound could be EPAtrans-esterified onto astaxanthin. Thus, to deliver the label amount ofeach carotenoid in a single dose, the krill oil and the carotenoids arenot allowed to mix because of the incompatibility issue. FIG. 1 showsthe technical solution using the capsule within a capsule in accordancewith a non-limiting example.

FIG. 1 shows the medicine delivery system 10 that includes the innercapsule 12 containing mixed carotenoids in one example comprising atleast S,S′-astaxanthin derived from Haematococcus pluvialis and one ormore of lutein and/or trans-zeaxanthin or meso-zeaxanthin. The outercapsule 14 contains a therapeutically effective amount of krill oilcomprising phospholipid bound and triglyceride bound EPA and DHA and thekrill oil contains at least 30% total phospholipids.

In an example, 50-1,000 mg of krill oil can be contained in the outercapsule 14. In another example, the mixed carotenoids can include 0.5 to8 mg of astaxanthin, 2 to 15 mg of lutein, and 0.2 to 12 mg oftrans-zeaxanthin within the inner capsule. About 4 mg of astaxanthin,about 10 of lutein and about 1.2 mg of trans-zeaxanthin is containedwithin the inner capsule in one example. The use of this medicinedelivery system 10 provides a new composition after ingestion in vivothat includes the therapeutically effective amount of synergisticmulti-ingredient composition of krill oil containing phospholipid boundand triglyceride bound EPA and DHA in which the krill oil contains atleast 30% total phospholipids and mixed carotenoids comprising at leastS,S′-astaxanthin derived from Haematococcus pluvialis that has reactedwith the krill oil, and one or more of lutein and/or trans-zeaxanthin ormeso-zeaxanthin. This allows a method of treating an individual byadministering the capsule that includes the therapeutically effectiveamount of a synergistic multi-ingredient composition of krill oil andcarotenoids and reacting carotenoids in vivo with the krill oil afteringestion.

FIG. 2 is a chart showing data of astaxanthin at 13 days postformulation reacting with krill oil, but essentially neither lutein norzeaxanthin. Astaxanthin is a keto-alcohol while lutein and zeaxanthinare alcohols and this can possibly make some difference. It should alsobe understood that the krill oil and “reacted” carotenoids, such asastaxanthin, and possibly zeaxanthin and lutein, lose their ability tobe recovered over time from the mixture but can still be quantitated byultraviolet (UV) analysis. For example, the chromophores of thecarotenoids are not destroyed when the reaction takes place over timeand can be used to develop a number of products useful forcardiovascular applications, joint health care applications, eye careand other diseases.

Referring now to the chart in FIG. 2, there are shown various samplesand explanation. Sample 3/1 is a mixture of astaxanthin in krill oilonly and showing a 23% reduction in HPLC recoverable astaxanthin by day13 while retaining total carotenoid concentration as measured by UVanalysis. Similar results were obtained in the presence of lutein,however the “loss” of recoverable lutein was minimal while astaxanthinprovided the same loss as the 3/1 sample. Sample 5/1 is mixedcarotenoids. In this example, there is essentially no loss of HPLCrecoverable astaxanthin while there is minimal loss of lutein andzeaxanthin (probably within experimental error). Recoveries of luteinand zeaxanthin in the presence of krill oil were within experimentalerror. Recoveries of these same carotenoids without krill oil, however,were less than expected because lutein and zeaxanthin are not verysoluble in astaxanthin oleoresin.

Admixture of astaxanthin derived from hp biomass extraction with krilloil above normal levels shows a continued loss of recoverableastaxanthin over day 1 concentrations while the total carotenoidconcentration as measured by UV analysis remains essentially unchanged,indicating that a chemical reaction is taking place when relatively highconcentrations of astaxanthin are added to krill oil. Further testingwith LC/MS/MS can determine the resulting chemical species being formed.It is believed this “reaction” is unique to krill oil phospholipids butother experiments can be conducted with other chemical species todetermine the scope of this reaction. Further determination can be madewhether all keto-alcohols (e.g., astaxanthin) react, and alcohols,amines, etc. A full strength eye care composition product included 4 mgof astaxanthin, 10 mg lutein and 1.2 mg zeaxanthin in an inner capsulewith krill oil in the outer capsule to prevent comingling of thecarotenoids with krill oil and preserve label claims in the event of anHPLC analysis of a simple blended product.

FIG. 3 is another chart showing a stability analytical report for theastaxanthin, lutein and zeaxanthin with percent change from day 0 to day12 at 20 degrees C. This is compared to FIG. 2 charts that show for day13. The charts in FIG. 2 show the Aker Biomarine sample while the chartsin FIG. 3 show use for an eye formula as claimed and also a FlexPro suchas disclosed in commonly assigned U.S. patent application Ser. No.12/840,372 and published as U.S. Patent Publication No. 2011/0020316with the A corresponding to Aker Biomarine krill oil and E correspondingto Enzymotec krill oil.

FIG. 4 is a chart showing results for 50 degrees C. FIG. 5 is a chartshowing summary of changes by UV as described above. FIG. 6 is anotherchart showing results with a day 26 added with the eye formulation asdescribed.

FIG. 7 shows the different chemical formula for different carotenoids.This indicates the differences and why krill oil can react more readilywith one type of carotenoid and not others.

Many modifications and other embodiments of the invention will come tothe mind of one skilled in the art having the benefit of the teachingspresented in the foregoing descriptions and the associated drawings.Therefore, it is understood that the invention is not to be limited tothe specific embodiments disclosed, and that modifications andembodiments are intended to be included within the scope of the appendedclaims.

1-20. (canceled)
 21. A method of treating an individual for aphysiological abnormality by administrating a therapeutically effectiveamount of a multi-ingredient composition of krill oil and carotenoidsand reacting the carotenoids in vivo with the krill oil.
 22. The methodaccording to claim 21, further comprising administering carotenoidscomprising at least S,S′-astaxanthin derived from Haematococcuspluvialis, and one or more of lutein and/or trans-zeaxanthin ormeso-zeaxanthin.
 23. The method according to claim 22, furthercomprising administering 0.5 to 8 mg of astaxanthin, 2 to 15 mg oflutein and 0.2 to 12 mg of trans-zeaxanthin.
 24. The method according toclaim 23, further comprising administering about 4 mg of astaxanthin,about 10 mg of lutein and about 1.2 mg of trans-zeaxanthin.
 25. Themethod according to claim 21, further comprising administering krill oilcontaining phospholipid bound and triglyceride bound EPA and DHA, thekrill oil comprising at least 30% total phospholipids.
 26. The methodaccording to claim 21, further comprising administering 50 to 1000 mg ofkrill oil.
 27. The method according to claim 21, further comprisingadministering carotenoids comprising astaxanthin.
 28. The methodaccording to claim 21, wherein the reacting carotenoids in vivo with thekrill oil comprises mixing krill oil contained within an outer capsulewith carotenoids contained within an inner capsule after capsuleingestion.
 29. A method of treating an individual for a physiologicalabnormality by administrating a therapeutically effective amount of amulti-ingredient composition of krill oil and astaxanthin and reactingthe astaxanthin in vivo with the krill oil.
 30. The method according toclaim 29, further comprising administering astaxanthin comprising atleast S,S′-astaxanthin derived from Haematococcus pluvialis.
 31. Themethod according to claim 29, further comprising administering one ormore of lutein and/or trans-zeaxanthin or meso-zeaxanthin.
 32. Themethod according to claim 31, further comprising administering 0.5 to 8mg of astaxanthin, 2 to 15 mg of lutein and 0.2 to 12 mg oftrans-zeaxanthin.
 33. The method according to claim 32, furthercomprising administering about 4 mg of astaxanthin, about 10 mg oflutein and about 1.2 mg of trans-zeaxanthin.
 34. The method according toclaim 29, further comprising administering krill oil containingphospholipid bound and triglyceride bound EPA and DHA, the krill oilcomprising at least 30% total phospholipids.
 35. The method according toclaim 29, further comprising administering 50 to 1000 mg of krill oil.36. The method according to claim 29, wherein the reacting astaxanthinin vivo with the krill oil comprises mixing krill oil contained withinan outer capsule with astaxanthin contained within an inner capsuleafter capsule ingestion.